Our state-of-the-art Single-Molecule RNA Fluorescence In Situ Hybridization (smRNA-FISH) service offers an indispensable approach to studying individual gene transcripts within single cells. This sophisticated technique involves the following key steps:
- Cell Cultivation: Cells of interest are cultured within multiwell plates (96 or 384 well) for defined periods, creating an environment conducive to detailed cellular exploration.
- Biochemical Investigations: We afford the flexibility to introduce a spectrum of biochemical interventions. This may involve the application of compounds of interest, thoughtfully aligned with the specific objectives of your research.
- Incubation and Fixation: Following the prescribed incubation period, the fixation process halts cellular processes, ensuring the molecular integrity of the specimens for subsequent analysis.
- Fluorescent DNA Probes: The core of smRNA FISH methodology involves the utilization of custom-designed, fluorescently labeled DNA oligonucleotide probes. The application of multiple probes per mRNA molecule as well as signal amplification methods significantly heightens the sensitivity of the assay, enabling the precise detection of mRNA expression. Our approach accommodates a dynamic range, from individual transcript molecules to several thousand, delivering a comprehensive view of gene expression at the single-cell level.
smRNA-FISH service represents a breakthrough in single-cell gene transcript analysis, shedding light on the intricacies of gene expression dynamics and contributing to a deeper understanding of cellular function and molecular processes. Notably, smRNA-FISH offers distinct advantages compared to traditional RNA-seq and qPCR methods:
- Spatial Mapping: Beyond mere quantification, our technique offers crucial insights into the spatial distribution of transcripts within the cell. This spatial context enriches our comprehension of gene function within distinct cellular locales.
- Multiplexing Capabilities: By fine-tuning the protocol for immunofluorescence staining, we enable the harmonious integration of smRNA-FISH with the concurrent detection of proteins of interest within the same single cell. This multifaceted approach unlocks the ability to scrutinize mRNA and protein expression simultaneously, uncovering intricate correlations between genetic and protein factors.
- Single-Cell Resolution: Unlike bulk RNA-seq techniques, smRNA-FISH provides single-cell resolution, allowing the examination of gene expression at the individual cell level which s crucial for identifying cell-to-cell variations and heterogeneity within a population.
- High Sensitivity: smRNA-FISH is highly sensitive and can detect low-abundance transcripts, making it a valuable tool for studying genes with low expression levels in single cells.
- Complementary Technique: smRNA-FISH can be used in conjunction with single-cell RNA-seq and qPCR to provide comprehensive insights into gene expression at different levels, offering a well-rounded approach to gene expression analysis.
Assay summary
| Stellaris RNA FISH | ViewRNA™ Cell Plus |
|
| Working principle | Method employs a set of 48 singly labelled oligonucleotides designed to selectively bind to targeted transcripts. Stellaris RNA FISH Probes bound to target RNA produce fluorescent signals that permit detection of single RNA molecules as diffraction-limited spots by conventional fluorescence microscopy. | Method employs branched DNA amplification technology to amplify signal detection of an RNA transcript. Specific set of approximately 20 short oligonucleotide pairs bind to the target RNA, and signal amplification occurs through a sequential hybridization process. |
| What is detected | Individual RNA molecules | Individual RNA molecules – signal amplified by increased number of labels associated with each target RNA molecule |
| Signal type | fluorescence | fluorescence |
| Platform | Automated fluorescence microscope | Automated fluorescence microscope |
| Sensitivity | 3/4 | 4/4 |
| Throughput | Medium | Medium |
| End-point/real time | End-point | End-point |
| Multiplexing | Up to three different transcripts | Up to three RNA targets simultaneously; a combination of one to two antibodies and a single RNA; or one antibody and two RNA |
| Model system | Adherent 2D cell cultures | Adherent 2D cell cultures |
Example output
